#METABOLOMICS WORKBENCH AbhaySatoskar_20231017_004228 DATATRACK_ID:4405 STUDY_ID:ST002974 ANALYSIS_ID:AN004883 PROJECT_ID:PR001851 VERSION 1 CREATED_ON November 14, 2023, 1:32 am #PROJECT PR:PROJECT_TITLE Leishmania mexicana Promotes Pain-reducing Metabolomic Reprogramming In PR:PROJECT_TITLE Cutaneous Lesions PR:PROJECT_TYPE Untargeted MS PR:PROJECT_SUMMARY Cutaneous leishmaniasis is characterized by extensive skin lesions, which are PR:PROJECT_SUMMARY usually painless despite being associated with extensive inflammation. The PR:PROJECT_SUMMARY molecular mechanisms responsible for this analgesia have not been identified. PR:PROJECT_SUMMARY Through untargeted metabolomics, we found enriched anti-nociceptive metabolic PR:PROJECT_SUMMARY pathways in L. mexicana-infected mice. Purines were elevated in infected PR:PROJECT_SUMMARY macrophages and at the lesion site during chronic infection. These purines have PR:PROJECT_SUMMARY anti-inflammatory and analgesic properties by acting through adenosine PR:PROJECT_SUMMARY receptors, inhibiting TRPV1 channels, and promoting IL-10 production. We also PR:PROJECT_SUMMARY found arachidonic acid metabolism enriched in the ear lesions compared to the PR:PROJECT_SUMMARY non-infected controls. Arachidonic acid is a metabolite of anandamide (AEA) and PR:PROJECT_SUMMARY 2-arachidonoylglycerol (2-AG). These endocannabinoids act on cannabinoid PR:PROJECT_SUMMARY receptors 1 and 2 and TRPV1 channels to exert anti-inflammatory and analgesic PR:PROJECT_SUMMARY effects. Our study provides evidence of metabolic pathways upregulated during L. PR:PROJECT_SUMMARY mexicana infection that may mediate anti-nociceptive effects experienced by CL PR:PROJECT_SUMMARY patients and identifies macrophages as a source of these metabolites. PR:INSTITUTE The Ohio State University PR:DEPARTMENT Pathology and Microbiology PR:LAST_NAME Satoskar PR:FIRST_NAME Abhay PR:ADDRESS Evans Hall, 520 King Avenue, Columbus, OH, 43201, USA PR:EMAIL abhay.satoskar@osumc.edu PR:PHONE (614) 293-0537 #STUDY ST:STUDY_TITLE Leishmania mexicana Promotes Pain-reducing Metabolomic Reprogramming In ST:STUDY_TITLE Cutaneous Lesions ST:STUDY_TYPE Untargeted MS ST:STUDY_SUMMARY Cutaneous leishmaniasis is characterized by extensive skin lesions, which are ST:STUDY_SUMMARY usually painless despite being associated with extensive inflammation. The ST:STUDY_SUMMARY molecular mechanisms responsible for this analgesia have not been identified. ST:STUDY_SUMMARY Through untargeted metabolomics, we found enriched anti-nociceptive metabolic ST:STUDY_SUMMARY pathways in L. mexicana-infected mice. Purines were elevated in infected ST:STUDY_SUMMARY macrophages and at the lesion site during chronic infection. These purines have ST:STUDY_SUMMARY anti-inflammatory and analgesic properties by acting through adenosine ST:STUDY_SUMMARY receptors, inhibiting TRPV1 channels, and promoting IL-10 production. We also ST:STUDY_SUMMARY found arachidonic acid metabolism enriched in the ear lesions compared to the ST:STUDY_SUMMARY non-infected controls. Arachidonic acid is a metabolite of anandamide (AEA) and ST:STUDY_SUMMARY 2-arachidonoylglycerol (2-AG). These endocannabinoids act on cannabinoid ST:STUDY_SUMMARY receptors 1 and 2 and TRPV1 channels to exert anti-inflammatory and analgesic ST:STUDY_SUMMARY effects. Our study provides evidence of metabolic pathways upregulated during L. ST:STUDY_SUMMARY mexicana infection that may mediate anti-nociceptive effects experienced by CL ST:STUDY_SUMMARY patients and identifies macrophages as a source of these metabolites. ST:INSTITUTE The Ohio State University ST:DEPARTMENT Pathology and Microbiology ST:LAST_NAME Satoskar ST:FIRST_NAME Abhay ST:ADDRESS Evans Hall, 520 King Avenue, Columbus, OH, 43201, USA ST:EMAIL abhay.satoskar@osumc.edu ST:PHONE (614) 293-0537 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Female #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - BMDMSup-Control1_1 Sample Type:Supernatant from naïve BMDMs. Biological replicate 1, technical replicate 1 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-Control1_2 Sample Type:Supernatant from naïve BMDMs. Biological replicate 1, technical replicate 2 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-Control1_3 Sample Type:Supernatant from naïve BMDMs. Biological replicate 1, technical replicate 3 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-Control2_1 Sample Type:Supernatant from naïve BMDMs. Biological replicate 2, technical replicate 1 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-Control2_2 Sample Type:Supernatant from naïve BMDMs. Biological replicate 2, technical replicate 2 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-Control2_3 Sample Type:Supernatant from naïve BMDMs. Biological replicate 2, technical replicate 3 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-Control3_1 Sample Type:Supernatant from naïve BMDMs. Biological replicate 3, technical replicate 1 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-Control3_2 Sample Type:Supernatant from naïve BMDMs. Biological replicate 3, technical replicate 2 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-Control3_3 Sample Type:Supernatant from naïve BMDMs. Biological replicate 3, technical replicate 3 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex1_1 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 1 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex1_2 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 2 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex1_3 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 3 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex2_1 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 1 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex2_2 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 2 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex2_3 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 3 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex3_1 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 1 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex3_2 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 2 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex3_3 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 3 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control1_1 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 1, technical replicate 1 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control1_2 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 1, technical replicate 2 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control1_3 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 1, technical replicate 3 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control2_1 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 2, technical replicate 1 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control2_2 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 2, technical replicate 2 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control2_3 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 2, technical replicate 3 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control3_1 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 3, technical replicate 1 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control3_2 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 3, technical replicate 2 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control3_3 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 3, technical replicate 3 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex1_1 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 1 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex1_2 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 2 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex1_3 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 3 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex2_1 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 1 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex2_2 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 2 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex2_3 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 3 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex3_1 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 1 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex3_2 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 2 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex3_3 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 3 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - VolpedoTissue_N1_1 Sample Type:Tissue from the ear of a naïve mouse. Biological replicate 1, technical replicate 1 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_N1_2 Sample Type:Tissue from the ear of a naïve mouse. Biological replicate 1, technical replicate 2 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_N1_3 Sample Type:Tissue from the ear of a naïve mouse. Biological replicate 1, technical replicate 3 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_N2_1 Sample Type:Tissue from the ear of a naïve mouse. Biological replicate 2, technical replicate 1 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_N2_2 Sample Type:Tissue from the ear of a naïve mouse. Biological replicate 2, technical replicate 2 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_N2_3 Sample Type:Tissue from the ear of a naïve mouse. Biological replicate 2, technical replicate 3 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi1_1 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 1, technical replicate 1 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi1_2 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 1, technical replicate 2 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi1_3 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 1, technical replicate 3 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi2_1 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 2, technical replicate 1 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi2_2 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 2, technical replicate 2 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi2_3 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 2, technical replicate 3 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi3_1 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 3, technical replicate 1 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi3_2 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 3, technical replicate 2 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi3_3 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 3, technical replicate 3 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi4_1 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 4, technical replicate 1 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi4_2 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 4, technical replicate 2 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi4_3 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 4, technical replicate 3 Mass spec mode=Positive mode & Negative mode SUBJECT_SAMPLE_FACTORS - RAW-L1_Cell_1 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 1, technical replicate 1 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - RAW-L1_Cell_2 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 1, technical replicate 2 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - RAW-L1_Cell_3 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 1, technical replicate 3 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - RAW-L2_Cell_1 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 2, technical replicate 1 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - RAW-L2_Cell_2 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 2, technical replicate 2 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - RAW-L2_Cell_3 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 2, technical replicate 3 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - RAW-L3_Cell_1 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 3, technical replicate 1 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - RAW-L3_Cell_2 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 3, technical replicate 2 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - RAW-L3_Cell_3 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 3, technical replicate 3 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - RAW-N_Cell_1 Sample Type:Cell pellet derived from naïve RAW 264.7. Technical replicate 1 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - RAW-N_Cell_2 Sample Type:Cell pellet derived from naïve RAW 264.7. Technical replicate 2 Mass spec mode=Positive SUBJECT_SAMPLE_FACTORS - RAW-N_Cell_3 Sample Type:Cell pellet derived from naïve RAW 264.7. Technical replicate 3 Mass spec mode=Positive #COLLECTION CO:COLLECTION_SUMMARY For in vivo studies, the ears were collected, snap frozen, and processed for CO:COLLECTION_SUMMARY mass spectrometry analysis. For in vitro experiments, the culture supernatant CO:COLLECTION_SUMMARY was collected and cell debris was removed by centrifugation. The attached cells CO:COLLECTION_SUMMARY were be scraped, washed with PBS and quenched with 80% methanol. Then they were CO:COLLECTION_SUMMARY snap-frozen, centrifuged, and lyophilized CO:COLLECTION_PROTOCOL_FILENAME Study_Methods-Volpedo_et_al.pdf CO:SAMPLE_TYPE Biopsy CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Experimental mice were infected intradermally in the ear with 1 million L. TR:TREATMENT_SUMMARY mexicana promastigotes. Naive mice received PBS injection. RAW 264.7 macrophages TR:TREATMENT_SUMMARY and BMDMs were plated in a 24-well plate at a density of 0.5x106 per well and TR:TREATMENT_SUMMARY infected overnight with stationary phase L. mexicana promastigotes at a ratio of TR:TREATMENT_SUMMARY 10:1 (parasite to macrophages). The controls were treated with media alone. TR:TREATMENT_SUMMARY Then, the extracellular parasites were removed by washing with PBS and new media TR:TREATMENT_SUMMARY was applied. After a 24hrs incubation, the supernatant and cell pellet were TR:TREATMENT_SUMMARY collected and processed for mass spectrometry. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For in vitro experiments, the culture supernatant was collected and cell debris SP:SAMPLEPREP_SUMMARY was removed by centrifugation according to SOP 5 of the Laboratory Guide for SP:SAMPLEPREP_SUMMARY Metabolomics Experiments. The attached cells were be scraped, washed with PBS SP:SAMPLEPREP_SUMMARY and quenched with 80% methanol. Then they were snap-frozen, centrifuged, and SP:SAMPLEPREP_SUMMARY lyophilized according to SOP 4 of the Laboratory Guide for Metabolomics SP:SAMPLEPREP_SUMMARY Experiments. For in vivo studies, the ears were collected, snap frozen, and SP:SAMPLEPREP_SUMMARY processed for mass spectrometry analysis according to SOP 7 of the Laboratory SP:SAMPLEPREP_SUMMARY Guide for Metabolomics Experiments. Samples were then incubated with 500 uL of SP:SAMPLEPREP_SUMMARY 100% MeOH and sonicated. The tissue was weighed and homogenized at 40 mg/mL of SP:SAMPLEPREP_SUMMARY 50% MeOH solution for 3 cycles in a Precellys homogenizer. The supernatant was SP:SAMPLEPREP_SUMMARY collected, dried down, and reconstituted in ½ of the original volume in 5 % SP:SAMPLEPREP_SUMMARY MeOH with 0.1 % formic acid. For metabolite candidates found via untargeted SP:SAMPLEPREP_SUMMARY analysis, pure standards purchased from Sigma-Aldrich were diluted in 100 % MeOH SP:SAMPLEPREP_SUMMARY stocks to 10 ug/mL, dried down, and reconstituted in 5 % MeOH with 0.1 % formic SP:SAMPLEPREP_SUMMARY acid and run in the same conditions described below to match features for SP:SAMPLEPREP_SUMMARY identification. Untargeted analysis was performed on a Thermo Scientific SP:SAMPLEPREP_SUMMARY Q-Exactive Plus Orbitrap mass spectrometer (MS) with HPLC separation on a SP:SAMPLEPREP_SUMMARY Poroshell 120 SB-C18 (2 x 100 mm, 2.7 µm particle size) with a Thermo SP:SAMPLEPREP_SUMMARY Scientific Ultimate WPS 3000 UHPLC system. The gradient consisted of solvent A, SP:SAMPLEPREP_SUMMARY H2O with 0.1% Formic acid, and solvent B 100% acetonitrile at a 200 µL/min flow SP:SAMPLEPREP_SUMMARY rate with an initial 2% solvent B with a linear ramp to 95% B at 15 min, holding SP:SAMPLEPREP_SUMMARY at 95% B for 1 minutes, and back to 5% B from 17 min and equilibration of 5% B SP:SAMPLEPREP_SUMMARY until min 30. For each sample, 5 µL was injected for each sample with SP:SAMPLEPREP_SUMMARY ionization in the MS on a HESI electrospray ionization source using a capillary SP:SAMPLEPREP_SUMMARY voltage of 4.5 kV in positive and 4.0 kV in negative mode at 320 °C capillary SP:SAMPLEPREP_SUMMARY temperature and 100 °C probe temperature. Gas settings were set to 15 for SP:SAMPLEPREP_SUMMARY sheath gas and 5 auxiliary gas while the S-Lens was set to 50 V. The top 5 ions SP:SAMPLEPREP_SUMMARY were selected for data dependent analysis with a 10 second exclusion window, SP:SAMPLEPREP_SUMMARY with a mass range of 80-1200 m/z and a resolution of 70,000 for MS scans and SP:SAMPLEPREP_SUMMARY 17,5000 for MSMS scans and fragmentation normalized collision energies of 30 V. SP:SAMPLEPREP_SUMMARY For feature selection in the untargeted results analysis, including database SP:SAMPLEPREP_SUMMARY comparison and statistical processing, samples were analyzed in Progenesis QI SP:SAMPLEPREP_SUMMARY and the pooled sample runs were selected for feature alignment. Using the pooled SP:SAMPLEPREP_SUMMARY QC samples, features with above 30% CV and max abundances below 5000 intensity SP:SAMPLEPREP_SUMMARY were filtered out and Anova p-value scores between the groups were calculated SP:SAMPLEPREP_SUMMARY with a cutoff of < 0.05. With database matching using the Human Metabolome SP:SAMPLEPREP_SUMMARY Database, selecting for adducts M+H, M+Na, M+2H, and 2M+H for positive mode and SP:SAMPLEPREP_SUMMARY M-H, M+Cl, M-2H, and 2M-H and less than 10 ppm mass error, unique features were SP:SAMPLEPREP_SUMMARY tentatively identified as potential metabolites. Metabolites were only annotated SP:SAMPLEPREP_SUMMARY with MSMS fragmentation matching scores above 20 % with Progenesis Metascope. SP:SAMPLEPREP_PROTOCOL_FILENAME Study_Methods-Volpedo_et_al.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Untargeted analysis was performed on a Thermo Scientific Q-Exactive Plus CH:CHROMATOGRAPHY_SUMMARY Orbitrap mass spectrometer (MS) with HPLC separation on a Poroshell 120 SB-C18 CH:CHROMATOGRAPHY_SUMMARY (2 x 100 mm, 2.7 µm particle size) with a Thermo Scientific Ultimate WPS 3000 CH:CHROMATOGRAPHY_SUMMARY UHPLC system. The gradient consisted of solvent A, H2O with 0.1% Formic acid, CH:CHROMATOGRAPHY_SUMMARY and solvent B 100% acetonitrile at a 200 µL/min flow rate with an initial 2% CH:CHROMATOGRAPHY_SUMMARY solvent B with a linear ramp to 95% B at 15 min, holding at 95% B for 1 minutes, CH:CHROMATOGRAPHY_SUMMARY and back to 5% B from 17 min and equilibration of 5% B until min 30. For each CH:CHROMATOGRAPHY_SUMMARY sample, 5 µL was injected for each sample with ionization in the MS on a HESI CH:CHROMATOGRAPHY_SUMMARY electrospray ionization source using a capillary voltage of 4.5 kV in positive CH:CHROMATOGRAPHY_SUMMARY and 4.0 kV in negative mode at 320 °C capillary temperature and 100 °C probe CH:CHROMATOGRAPHY_SUMMARY temperature. Gas settings were set to 15 for sheath gas and 5 auxiliary gas CH:CHROMATOGRAPHY_SUMMARY while the S-Lens was set to 50 V. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Scientific Ultimate WPS 3000 UHPLC system CH:COLUMN_NAME Agilent Infinity Poroshell 120 SB-C18 (100 x 2 mm,2.7 µm) CH:SOLVENT_A H2O with 0.1% Formic acid CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT an initial 2% solvent B with a linear ramp to 95% B at 15 min, holding at 95% B CH:FLOW_GRADIENT for 1 minutes, and back to 5% B from 17 min and equilibration of 5% B until min CH:FLOW_GRADIENT 30 CH:FLOW_RATE 200 µL/min flow rate CH:COLUMN_TEMPERATURE 320 °C capillary temperature and 100 °C probe temperature CH:METHODS_FILENAME Study_Methods-Volpedo_et_al.pdf #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE Study_Methods-Volpedo_et_al.pdf #MS MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS See Study Methods for full procedures. After LC-MS run, the top 5 ions were MS:MS_COMMENTS selected for data dependent analysis with a 10 second exclusion window, with a MS:MS_COMMENTS mass range of 80-1200 m/z and a resolution of 70,000 for MS scans and 17,5000 MS:MS_COMMENTS for MSMS scans and fragmentation normalized collision energies of 30 V. For MS:MS_COMMENTS feature selection in the untargeted results analysis, including database MS:MS_COMMENTS comparison and statistical processing, samples were analyzed in Progenesis QI MS:MS_COMMENTS and the pooled sample runs were selected for feature alignment. Using the pooled MS:MS_COMMENTS QC samples, features with above 30% CV and max abundances below 5000 intensity MS:MS_COMMENTS were filtered out and Anova p-value scores between the groups were calculated MS:MS_COMMENTS with a cutoff of < 0.05. With database matching using the Human Metabolome MS:MS_COMMENTS Database, selecting for adducts M+H, M+Na, M+2H, and 2M+H for positive mode and MS:MS_COMMENTS M-H, M+Cl, M-2H, and 2M-H and less than 10 ppm mass error, unique features were MS:MS_COMMENTS tentatively identified as potential metabolites. Metabolites were only annotated MS:MS_COMMENTS with MSMS fragmentation matching scores above 20 % with Progenesis Metascope. MS:MS_RESULTS_FILE ST002974_AN004883_Results.txt UNITS:ppm Has m/z:Yes Has RT:Yes RT units:Minutes #END